Project Summary The research proposed in this application is to investigate a novel crosstalk between Anaplastic Lymphoma Kinase (ALK) and TGF-? signaling pathway and the significance of this crosstalk in guiding the treatment of ALK-positive lung cancer. TGF-? signaling represents a major tumor suppressor pathway. Loss of the TGF-? response is a hallmark in human cancer. TGF-? inhibits cell proliferation through Smad-dependent transcription mechanisms. Smads, especially the tumor suppressor Smad4/DPC4, are frequently inactivated in many human malignancies. However, the mechanisms underlying TGF-? resistance have not been fully elucidated in cancers, particularly in those (including lung cancer) where there are no deletions or mutations in the genes encoding Smad4 or other components in the TGF-? pathway. Moreover, in late stages of tumor progression when tumor cells become resistant to growth inhibition by TGF-? due to inactivation of the TGF-? signaling pathway or aberrant regulation of the cell cycle, the role of TGF-? becomes one of tumor promoters as TGF-? enhances EMT, cell migration/ invasion and cancer metastasis. Thus, the importance of understanding the function and regulation of TGF-? activity can never be overstated. Anaplastic Lymphoma Kinase (ALK) is a member of the insulin receptor tyrosine kinase superfamily. Gene fusion, mutations, amplification and aberrant expression of ALK have been reported in multiple types of human cancer including lung cancer. The molecular mechanism underlying ALK oncogenesis, which can act through the activation of Ras-MAPK and PI3K pathways, remains only partially understood. In our preliminary studies with ALK positive cancer cell lines, we have for the first time discovered that ALK could inactivate Smad4 tumor suppressive functions by a novel tyrosine phosphorylation. We found: (1) Smad4 is tyrosine- phosphorylated by ALK, and this tyrosine-phosphorylated Smad4 has decreased DNA-binding ability and fails to mediate TGF-? signal transduction; (2) expression of constitutively active ALK (caALK) in ALK-negative, TGF-?-responsive cells attenuates TGF-?-induced gene expression; and (3) depletion of ALK restores TGF-? transcriptional and growth inhibitory responses in ALK-positive, TGF-?-resistant cells. In this proposal, we hypothesize that ALK disrupts TGF-? signaling through Smad4 inactivation and thus affects tumorigenesis and progression. Because TGF-? signaling can both suppress early tumor formation and promote progression of clinically diagnosed tumors, the consequence of using an ALK inhibitor such as crizotinib to treat established cancer will not only inactivate ALK signaling, but also likely restore TGF-? signaling, which can promote cancer spread. To test this hypothesis, we will leverage our rich basic and clinical resource and animal models in lung cancer. Specifically, we will use cell-based assays and in vivo lung cancer mouse model as well as human lung cancer specimens to determine whether aberrant activation of ALK causes TGF-? resistance by Smad4 tyrosine phosphorylation and inactivation. Furthermore, we will determine the impact of ALK activation on the tumor suppressor activity of Smad4, lung tumor initiation and progression using human tissues and mouse models, and to investigate the effects of ALK and TGF-? inhibitor on lung cancer formation and metastasis in mouse models. Thus, our proposed research is significant in that our proposed experiments will expose potential harm of ALK inhibitors in treating established cancer and may help or guide the ongoing clinical trials on lung cancer and other ALK-positive human tumors. In addition, since we have developed a sensitive immunohistochemical method for detecting Smad4 tyrosine phosphorylation in a clinical setting, our proposed research may identify phosphor-Smad4 as a diagnostic marker for ALK-positive cancers in a clinical setting or as a prognosis or predictive marker.